We have been studying the molecular genetic mechanisms that control the development switch from embryonic to fetal adult hemoglobins with respect to their basic biology and their relevance to developing new therapies for diseases of hemoglobin. To study these processes we have been using two model systems: the K562 erythroleukemic cells and transgenic mice. K562 is an erythroleukemic cell line used for the last decade as a model for the study of the control of the human globin gene expression. These cells do not support transcription of beta-globin gene but do express transcripts of epsilon and gamma-globin genes at a very high levels when exposed to a number of inducing agents. Results from this and other laboratories suggest that the control of this pattern of expression is mediated by the presence and/or absence trans-acting factors which exert their action on sequences corresponding to the promoters of these genes. In the last few years transgenic mice have become an excellent model system for studying globin gene expression, and are supplementing the use of human cell lines. We have previously reported the presence of a transcriptional control element with properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin gene. Using deletion mutants and synthetic oligonucleotides in transient expression assays, DNA sequences responsible for this effect have been further delimited to 44 nucleotides located between -294 and -251bp. Gel electrophoresis mobility shift assays and DNase footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. The protein present in K562 cells, but not in HeLa cells, that interacts specifically with this silencer binds to the same sequence to study the epsilon-globin silencer; constructs with the silencer sequence intact, as well as constructs with the silencer mutated, have been injected into fertilized mouse ova to study the function of the silencer in this assay system. The results should help clarify the molecular mechanisms of silencing, an important aspect of hemoglobin study.